High-resolution low-cost LCD 3D printing for microfluidics and organ-on-a-chip devices.

The fabrication of microfluidic devices has progressed from cleanroom manufacturing to replica molding in polymers, and more recently to direct manufacturing by subtractive (, laser machining) and additive (, 3D printing) techniques, notably digital light processing (DLP) photopolymerization. However, many methods require technical expertise and DLP 3D printers remain expensive at a cost ∼15-30 K USD with ∼8 M pixels that are 25-40 μm in size. Here, we introduce (i) the use of low-cost (∼150-600 USD) liquid crystal display (LCD) photopolymerization 3D printing with ∼8-58 M pixels that are 18-35 μm in size for direct microfluidic device fabrication, and (ii) a poly(ethylene glycol) diacrylate-based ink developed for LCD 3D printing (PLInk).

Viscoelastic hydrogels regulate adipose-derived mesenchymal stem cells for nucleus pulposus regeneration.

Low back pain is a leading cause of disability worldwide, often attributed to intervertebral disc (IVD) degeneration with loss of the functional nucleus pulposus (NP). Regenerative strategies utilizing biomaterials and stem cells are promising for NP repair. Human NP tissue is highly viscoelastic, relaxing stress rapidly under deformation.

Effect of Sample Preprocessing and Size-Based Extraction Methods on the Physical and Molecular Profiles of Extracellular Vesicles.

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted, and often preconcentrated. Here we first compare plasma preservation agents, and second, using both plasma and cell supernatant, four EV extraction methods, including (i) ultracentrifugation (UC), (ii) size-exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS).

High-Resolution Additive Manufacturing of a Biodegradable Elastomer with A Low-Cost LCD 3D Printer.

Artificial organs and organs-on-a-chip (OoC) are of great clinical and scientific interest and have recently been made by additive manufacturing, but depend on, and benefit from, biocompatible, biodegradable, and soft materials. Poly(octamethylene maleate (anhydride) citrate (POMaC) meets these criteria and has gained popularity, and as in principle, it can be photocured and is amenable to vat-photopolymerization (VP) 3D printing, but only low-resolution structures have been produced so far. Here, a VP-POMaC ink is introduced and 3D printing of 80 µm positive features and complex 3D structures is demonstrated using low-cost (≈US$300) liquid-crystal display (LCD) printers.

Digital Manufacturing of Functional Ready-to-Use Microfluidic Systems.

Digital manufacturing (DM) holds great potential for microfluidics, but requirements for embedded conduits and high resolution beyond the capability of common manufacturing equipment, and microfluidic systems' dependence on peripheralshave limited its adoption. Capillaric circuits (CCs) are structurally encoded, self-contained microfluidic systems that operate and self-fill via precisely tailored hydrophilicity. CCs  are  heretofore hydrophilized in a plasma chamber, but which offers only transient hydrophilicity, lacks reproducibility, and limits CC design to open surface channels subsequently sealed with tape.

nELISA: A high-throughput, high-plex platform enables quantitative profiling of the secretome.

We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry.

3D-printed capillaric ELISA-on-a-chip with aliquoting.

Sandwich immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have been miniaturized and performed in a lab-on-a-chip format, but the execution of the multiple assay steps typically requires a computer or complex peripherals. Recently, an ELISA for detecting antibodies was encoded structurally in a chip thanks to the microfluidic chain reaction (Yafia , 2022, , 464-469), but the need for precise pipetting and intolerance to commonly used surfactant concentrations limit the potential for broader adoption. Here, we introduce the ELISA-on-a-chip with aliquoting functionality that simplifies chip loading and pipetting, accommodates higher surfactant concentrations, includes barrier channels that delay the contact between solutions and prevent undesired mixing, and that executed a quantitative, high-sensitivity assay for the SARS-CoV-2 nucleocapsid protein in 4×-diluted saliva.

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis.

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging.

The Mini Colon Model: a benchtop multi-bioreactor system to investigate the gut microbiome.

fermentation systems allow for the investigation of gut microbial communities with precise control of various physiological parameters while decoupling confounding factors from the human host. Current systems, such as the SHIME and Robogut, are large in footprint, lack multiplexing, and have low experimental throughput. Alternatives which address these shortcomings, such as the Mini Bioreactor Array system, are often reliant on expensive specialized equipment, which hinders wide replication across labs.

Microfluidic chain reaction of structurally programmed capillary flow events.

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system) was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation.

Emerging Technologies in Multi-Material Bioprinting.

Bioprinting, within the emerging field of biofabrication, aims at the fabrication of functional biomimetic constructs. Different 3D bioprinting techniques have been adapted to bioprint cell-laden bioinks. However, single-material bioprinting techniques oftentimes fail to reproduce the complex compositions and diversity of native tissues.

Spatial Bias in Antibody Microarrays May Be an Underappreciated Source of Variability.

Antibody microarrays enable multiplexed protein detection with minimal reagent consumption, but they continue to be plagued by lack of reproducibility. Chemically functionalized glass slides are used as substrates, yet antibody binding spatial inhomogeneity across the slide has not been analyzed in antibody microarrays. Here, we characterize spatial bias across five commercial slides patterned with nine overlapping dense arrays (by combining three buffers and three different antibodies), and we measure signal variation for both antibody immobilization and the assay signal, generating 270 heatmaps.

Combinatorial nanodot stripe assay to systematically study cell haptotaxis.

Haptotaxis is critical to cell guidance and development and has been studied in vitro using either gradients or stripe assays that present a binary choice between full and zero coverage of a protein cue. However, stripes offer only a choice between extremes, while for gradients, cell receptor saturation, migration history, and directional persistence confound the interpretation of cellular responses. Here, we introduce nanodot stripe assays (NSAs) formed by adjacent stripes of nanodot arrays with different surface coverage.

Precise Chip-to-Chip Reagent Transfer for Cross-Reactivity-Free Multiplex Sandwich Immunoassays.

Common multiplex sandwich immunoassays suffer from cross-reactivity due to the mixing of detection antibodies and the combinatorial, undesired interaction between all reagents and analytes. Here we present the snap chip to perform antibody colocalization microarrays that eliminates undesirable interactions by running an array of singleplex assays realized by sequestering detection antibodies in individual nanodroplets. When detecting proteins in biological fluids, the absence of cross-reactivity allows a higher level of multiplexing, reduced background, increased sensitivity, and ensures accurate and specific results.

Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8 T cell activation in fluorinated ethylene propylene cell culture bags.

Background: A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags.

Microfluidic multipoles theory and applications.

Microfluidic multipoles (MFMs) have been realized experimentally and hold promise for "open-space" biological and chemical surface processing. Whereas convective flow can readily be predicted using hydraulic-electrical analogies, the design of advanced microfluidic multipole is constrained by the lack of simple, accurate models to predict mass transport within them. In this work, we introduce the complete solutions to mass transport in multipolar microfluidics based on the iterative conformal mapping of 2D advection-diffusion around a simple edge into dipoles and multipolar geometries, revealing a rich landscape of transport modes.

Hydrogel droplet single-cell processing: DNA purification, handling, release, and on-chip linearization.

The preparation and handling of mammalian single-cell genomic DNA is limited by the complexity bottleneck inherent to performing multi-step, multi-reagent operations in a microfluidic environment. We have developed a method for benchtop preparation of high-molecular weight, intact, single-cell genomes and demonstrate the extraction of long nucleic acid molecules in a microfluidic system. Lymphoblasts are encapsulated inside of alginate microparticles using a droplet microfluidics, and cells are lysed in bulk.

Duplexed aptamers: history, design, theory, and application to biosensing.

Nucleic acid aptamers are single stranded DNA or RNA sequences that specifically bind a cognate ligand. In addition to their widespread use as stand-alone affinity binding reagents in analytical chemistry, aptamers have been engineered into a variety of ligand-specific biosensors, termed aptasensors. One of the most common aptasensor formats is the duplexed aptamer (DA).

Two-level submicron high porosity membranes (2LHPM) for the capture and release of white blood cells (WBCs).

A method modifying a vacuum-assisted UV micro-molding (VAUM) process is proposed for the fabrication of polymer two-level submicron high porosity membranes (2LHPM). The modified process allows for the fabrication of robust, large-area membranes over 5 × 5 cm2 with a hierarchical architecture made from a 200 nm-thick layer having submicron level pores (as small as 500 nm) supported by a 20 μm-thick layer forming a microporous structure with 10-15 μm diameter pores. The fabricated freestanding membranes are flexible and mechanically robust enough for post manipulation and filtration of cell samples.

Protein microarray spots are modulated by patterning method, surface chemistry and processing conditions.

The uniformity of the protein patterns, their shape, and the contrast between the fluorescence signal of the pattern and the background, critically modulate the quantitative accuracy of the microarray-derived data. While significant research focused of the identification of the factors that impact the protein microarray patterns, these studies usually have focused on the optimization of one set of these factors, e.g.

Ensemble multicolour FRET model enables barcoding at extreme FRET levels.

Quantitative models of Förster resonance energy transfer (FRET)-pioneered by Förster-define our understanding of FRET and underpin its widespread use. However, multicolour FRET (mFRET), which arises between multiple, stochastically distributed fluorophores, lacks a mechanistic model and remains intractable. mFRET notably arises in fluorescently barcoded microparticles, resulting in a complex, non-orthogonal fluorescence response that impedes their encoding and decoding.

Capillary microfluidics in microchannels: from microfluidic networks to capillaric circuits.

Microfluidics offer economy of reagents, rapid liquid delivery, and potential for automation of many reactions, but often require peripheral equipment for flow control. Capillary microfluidics can deliver liquids in a pre-programmed manner without peripheral equipment by exploiting surface tension effects encoded by the geometry and surface chemistry of a microchannel. Here, we review the history and progress of microchannel-based capillary microfluidics spanning over three decades.

Comprehensive profiling of the ligand binding landscapes of duplexed aptamer families reveals widespread induced fit.

Duplexed aptamers (DAs) are ligand-responsive constructs engineered by hybridizing an aptamer with an aptamer-complementary element (ACE, e.g., a DNA oligonucleotide).

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities.